Structure probing method comparison¶
Here we compare the results of four studies that reported full-length SARS-CoV-2 secondary structures based on in-cell structure probing data.
In short, cells infected with SARS-CoV-2 were probed with a structure probing reagent. Then, sites of chemical adducts were encoded in cDNA libaries and sequenced. Chemical reactivity is estimated using a bioinformatic pipeline. Structure prediction is performed using chemical reactivity as restraints. All four studies differed in the details of each of these steps. Refer to Table S1 for the differences between these experiments.
For each publication, per-nucleotide reactivities and secondary structure models were downloaded from the original publications with one exception. The full-length structure model from Sun et al. was not attached in the original publication, but was found in the supplemental data from Lan et al.
In addition, we performed our own structure modelling, using RNAstructure Fold, of the first 8000 nucleotides of SARS-CoV-2, with a max pairing distance of 300 (--md 300) and without chemical probing data.
Our de novo structure model:
- Files and commands (RNAvigate_figures/interactive_notebooks/figure_6/denovo_model)
- Structure model file
Manfredonia et al. 2020 link
- Data set website link
- Reactivity data file download
- Structure model file download
Sun et al. 2021 link
- Reactivity data file download
Lan et al. 2022 link
- Structure model file download
- Reactivity data file download
- Supplemental data file containing Sun et al. structure model file download
Huston et al. 2021 link
- Github repo link
- Reactivity data file download
- Structure model file download
Import RNAvigate¶
The first step in this notebook is to import the necessary Python modules: RNAvigate and Pandas. Pandas is needed to load Excel files into RNAvigate.
import rnavigate as rnav
import pandas as pd
# this prevents wordwrap, which we'll need later to inspect a very long sequence.
from IPython.core.display import display, HTML
display(HTML("<style>div.output_area pre {white-space: pre;}</style>"))
Define samples and provide input file names¶
Next, data files are loaded into RNAvigate samples, and assigned data keywords.
shapemapstores SHAPE-MaP, icSHAPE, or DMS-MaPseq data depending on the study.ssstores the secondary structure model from each study.
We create one sample for each study (lan, huston, manfredonia, and sun),
and one sample to hold our no-data model (de_novo).
RNAvigate does not natively accept Excel files. Instead, Pandas is used to create DataFrames, which are accepted, from the Excel files. This requires only one extra python function.
de_novo = rnav.Sample(
sample="1-8000, max distance 300",
ss="./denovo_model/sars_1_8000_md300.ct")
huston = rnav.Sample(
sample="Huston et al.",
shapemap="./Huston_etal_2021/SARS-CoV-2_SHAPE_Reactivity.map",
ss="./Huston_etal_2021/SARS-CoV-2_Full_Length_Secondary_Structure_Map.ct")
manfredonia = rnav.Sample(
sample="Manfredonia et al.",
shapemap={
"shapemap_rnaframework": "./Manfredonia_etal_2020/SHAPE_invivo/SHAPE_invivo.xml"
},
ss="./Manfredonia_etal_2020/SHAPE_invivo/SARS-CoV-2.db")
lan_profile_df = pd.read_excel(
io="./Lan_etal_2022/population_reactivities.xlsx",
sheet_name="mus",
usecols=[0, 1, 3],
names=["Nucleotide", "Sequence", "Norm_profile"])
lan = rnav.Sample(
sample="Lan et al.",
shapemap=lan_profile_df,
ss="./Lan_etal_2022/vero.ct")
sun_profile_df = pd.read_excel(
io="./Sun_etal_2021/mmc2.xlsx",
sheet_name="SARS2-invivo",
usecols=[0, 1, 2],
names=["Nucleotide", "Sequence", "Norm_profile"],
dtype={
"Nucleotide": "Int32",
"Sequence": "string",
"Norm_profile": "float32"}
)
sun_ss_df = pd.read_excel(
io="./Lan_etal_2022/all_figure_data.xlsx",
sheet_name="Figure S5",
usecols=[0, 13],
names=["Nucleotide", "Pair"],
nrows=21289,
skiprows=5,
dtype={
"Nucleotide": "Int32",
"Pair": "Int32"})
pairs_list = [(nt, pair_nt) for _, (nt, pair_nt) in sun_ss_df.iterrows()]
sun = rnav.Sample(
sample="Sun et al.",
shapemap=sun_profile_df,
ss={'ss_pairs': pairs_list, 'sequence': 'shapemap'}
)
Making some changes to the data¶
Some of the loaded file formats specify DNA alphabet sequences and may contain lowercase letters. Here, all associated sequences are converted to an uppercase RNA alphabet.
Lan et al. profile data were provided as raw mutation rates. Here, DMS data are renormalized with a standard mutation rate correction for DMS-MaP data.
# Make sure that all sequences are RNA alphabet and uppercase
for sample in [lan, huston, manfredonia, sun, de_novo]:
for data_keyword in ['shapemap', 'ss']:
if data_keyword not in sample.data:
continue
sample.get_data(data_keyword).normalize_sequence(
t_or_u='U',
uppercase=True,
)
# Perform DMS normalization for DMS samples
lan.get_data('shapemap').normalize(
norm_method='percentiles',
nt_groups=['A','C']
);
Warning: Lan et al. (shapemap) missing expected error column
Check sequence alignments¶
We don't know what base sequences are being used, and if they differ positionally from each other. RNAvigate's SequenceChecker analysis can be used to display this information. In this first step, SequenceChecker is used to write a fasta file for input into ClustalOmega (link) to obtain a multiple sequence alignment.
seq_check = rnav.analysis.SequenceChecker(samples=[lan, huston, manfredonia, sun, de_novo])
seq_check.write_fasta('sequences.fa')
Check sequence alignments (continued)¶
In this next step, the sequence alignment file from the previous step is loaded into RNAvigate using rnav.data.set_multiple_sequence_alignment.
Whenever data is compared between these samples, they are first positionally aligned using this alignment.
RNAvigate can perform it's own pairwise alignment, but it is computationally expensive for this 29 kb RNA sequence.
Then, the SequenceChecker analysis created in the previous step can display all samples and data keywords and which sequences they contain, as well as the multiple sequence alignment.
base_seq = rnav.data.set_multiple_sequence_alignment('alignments.fa', set_pairwise=True)
seq_check.print_which_sequences()
seq_check.print_mulitple_sequence_alignment(base_seq)
Sequence IDs
Lan et al.
ss 0
shapemap 0.0
Huston et al.
ss 1
shapemap 1.0
Manfredonia et al.
ss 2
shapemap 3.0
Sun et al.
ss 1
shapemap 1.0
1-8000, max distance 300
ss 4
Multiple sequence alignment
ID length alignment
0 29882 AUUAAAGGUUUAUACCUUCCCAGGUAACAAACCAACCAACUUUCGAUCUCUUGUAGAUCUGUUCUCUAAACGAACUUUAAAAUCUGUGUGGCUGUCACUCGGCUGCAUGCUUAGUGCACUCACGCAGUAUAAUUAAUAACUAAUUACUGUCGUUGACAGGACACGAGUAACUCGUCUAUCUUCUGCAGGCUGCUUACGGUUUCGUCCGUGUUGCAGCCGAUCAUCAGCACAUCUAGGUUUCGUCCGGGUGUGACCGAAAGGUAAGAUGGAGAGCCUUGUCCCUGGUUUCAACGAGAAAACACACGUCCAACUCAGUUUGCCUGUUUUACAGGUUCGCGACGUGCUCGUACGUGGCUUUGGAGACUCCGUGGAGGAGGUCUUAUCAGAGGCACGUCAACAUCUUAAAGAUGGCACUUGUGGCUUAGUAGAAGUUGAAAAAGGCGUUUUGCCUCAACUUGAACAGCCCUAUGUGUUCAUCAAACGUUCGGAUGCUCGAACUGCACCUCAUGGUCAUGUUAUGGUUGAGCUGGUAGCAGAACUCGAAGGCAUUCAGUACGGUCGUAGUGGUGAGACACUUGGUGUCCUUGUCCCUCAUGUGGGCGAAAUACCAGUGGCUUACCGCAAGGUUCUUCUUCGUAAGAACGGUAAUAAAGGAGCUGGUGGCCAUAGUUACGGCGCCGAUCUAAAGUCAUUUGACUUAGGCGACGAGCUUGGCACUGAUCCUUAUGAAGAUUUUCAAGAAAACUGGAACACUAAACAUAGCAGUGGUGUUACCCGUGAACUCAUGCGUGAGCUUAACGGAGGGGCAUACACUCGCUAUGUCGAUAACAACUUCUGUGGCCCUGAUGGCUACCCUCUUGAGUGCAUUAAAGACCUUCUAGCACGUGCUGGUAAAGCUUCAUGCACUUUGUCCGAACAACUGGACUUUAUUGACACUAAGAGGGGUGUAUACUGCUGCCGUGAACAUGAGCAUGAAAUUGCUUGGUACACGGAACGUUCUGAAAAGAGCUAUGAAUUGCAGACACCUUUUGAAAUUAAAUUGGCAAAGAAAUUUGACACCUUCAAUGGGGAAUGUCCAAAUUUUGUAUUUCCCUUAAAUUCCAUAAUCAAGACUAUUCAACCAAGGGUUGAAAAGAAAAAGCUUGAUGGCUUUAUGGGUAGAAUUCGAUCUGUCUAUCCAGUUGCGUCACCAAAUGAAUGCAACCAAAUGUGCCUUUCAACUCUCAUGAAGUGUGAUCAUUGUGGUGAAACUUCAUGGCAGACGGGCGAUUUUGUUAAAGCCACUUGCGAAUUUUGUGGCACUGAGAAUUUGACUAAAGAAGGUGCCACUACUUGUGGUUACUUACCCCAAAAUGCUGUUGUUAAAAUUUAUUGUCCAGCAUGUCACAAUUCAGAAGUAGGACCUGAGCAUAGUCUUGCCGAAUACCAUAAUGAAUCUGGCUUGAAAACCAUUCUUCGUAAGGGUGGUCGCACUAUUGCCUUUGGAGGCUGUGUGUUCUCUUAUGUUGGUUGCCAUAACAAGUGUGCCUAUUGGGUUCCACGUGCUAGCGCUAACAUAGGUUGUAACCAUACAGGUGUUGUUGGAGAAGGUUCCGAAGGUCUUAAUGACAACCUUCUUGAAAUACUCCAAAAAGAGAAAGUCAACAUCAAUAUUGUUGGUGACUUUAAACUUAAUGAAGAGAUCGCCAUUAUUUUGGCAUCUUUUUCUGCUUCCACAAGUGCUUUUGUGGAAACUGUGAAAGGUUUGGAUUAUAAAGCAUUCAAACAAAUUGUUGAAUCCUGUGGUAAUUUUAAAGUUACAAAAGGAAAAGCUAAAAAAGGUGCCUGGAAUAUUGGUGAACAGAAAUCAAUACUGAGUCCUCUUUAUGCAUUUGCAUCAGAGGCUGCUCGUGUUGUACGAUCAAUUUUCUCCCGCACUCUUGAAACUGCUCAAAAUUCUGUGCGUGUUUUACAGAAGGCCGCUAUAACAAUACUAGAUGGAAUUUCACAGUAUUCACUGAGACUCAUUGAUGCUAUGAUGUUCACAUCUGAUUUGGCUACUAACAAUCUAGUUGUAAUGGCCUACAUUACAGGUGGUGUUGUUCAGUUGACUUCGCAGUGGCUAACUAACAUCUUUGGCACUGUUUAUGAAAAACUCAAACCCGUCCUUGAUUGGCUUGAAGAGAAGUUUAAGGAAGGUGUAGAGUUUCUUAGAGACGGUUGGGAAAUUGUUAAAUUUAUCUCAACCUGUGCUUGUGAAAUUGUCGGUGGACAAAUUGUCACCUGUGCAAAGGAAAUUAAGGAGAGUGUUCAGACAUUCUUUAAGCUUGUAAAUAAAUUUUUGGCUUUGUGUGCUGACUCUAUCAUUAUUGGUGGAGCUAAACUUAAAGCCUUGAAUUUAGGUGAAACAUUUGUCACGCACUCAAAGGGAUUGUACAGAAAGUGUGUUAAAUCCAGAGAAGAAACUGGCCUACUCAUGCCUCUAAAAGCCCCAAAAGAAAUUAUCUUCUUAGAGGGAGAAACACUUCCCACAGAAGUGUUAACAGAGGAAGUUGUCUUGAAAACUGGUGAUUUACAACCAUUAGAACAACCUACUAGUGAAGCUGUUGAAGCUCCAUUGGUUGGUACACCAGUUUGUAUUAACGGGCUUAUGUUGCUCGAAAUCAAAGACACAGAAAAGUACUGUGCCCUUGCACCUAAUAUGAUGGUAACAAACAAUACCUUCACACUCAAAGGCGGUGCACCAACAAAGGUUACUUUUGGUGAUGACACUGUGAUAGAAGUGCAAGGUUACAAGAGUGUGAAUAUCACUUUUGAACUUGAUGAAAGGAUUGAUAAAGUACUUAAUGAGAAGUGCUCUGCCUAUACAGUUGAACUCGGUACAGAAGUAAAUGAGUUCGCCUGUGUUGUGGCAGAUGCUGUCAUAAAAACUUUGCAACCAGUAUCUGAAUUACUUACACCACUGGGCAUUGAUUUAGAUGAGUGGAGUAUGGCUACAUACUACUUAUUUGAUGAGUCUGGUGAGUUUAAAUUGGCUUCACAUAUGUAUUGUUCUUUCUACCCUCCAGAUGAGGAUGAAGAAGAAGGUGAUUGUGAAGAAGAAGAGUUUGAGCCAUCAACUCAAUAUGAGUAUGGUACUGAAGAUGAUUACCAAGGUAAACCUUUGGAAUUUGGUGCCACUUCUGCUGCUCUUCAACCUGAAGAAGAGCAAGAAGAAGAUUGGUUAGAUGAUGAUAGUCAACAAACUGUUGGUCAACAAGACGGCAGUGAGGACAAUCAGACAACUACUAUUCAAACAAUUGUUGAGGUUCAACCUCAAUUAGAGAUGGAACUUACACCAGUUGUUCAGACUAUUGAAGUGAAUAGUUUUAGUGGUUAUUUAAAACUUACUGACAAUGUAUACAUUAAAAAUGCAGACAUUGUGGAAGAAGCUAAAAAGGUAAAACCAACAGUGGUUGUUAAUGCAGCCAAUGUUUACCUUAAACAUGGAGGAGGUGUUGCAGGAGCCUUAAAUAAGGCUACUAACAAUGCCAUGCAAGUUGAAUCUGAUGAUUACAUAGCUACUAAUGGACCACUUAAAGUGGGUGGUAGUUGUGUUUUAAGCGGACACAAUCUUGCUAAACACUGUCUUCAUGUUGUCGGCCCAAAUGUUAACAAAGGUGAAGACAUUCAACUUCUUAAGAGUGCUUAUGAAAAUUUUAAUCAGCACGAAGUUCUACUUGCACCAUUAUUAUCAGCUGGUAUUUUUGGUGCUGACCCUAUACAUUCUUUAAGAGUUUGUGUAGAUACUGUUCGCACAAAUGUCUACUUAGCUGUCUUUGAUAAAAAUCUCUAUGACAAACUUGUUUCAAGCUUUUUGGAAAUGAAGAGUGAAAAGCAAGUUGAACAAAAGAUCGCUGAGAUUCCUAAAGAGGAAGUUAAGCCAUUUAUAACUGAAAGUAAACCUUCAGUUGAACAGAGAAAACAAGAUGAUAAGAAAAUCAAAGCUUGUGUUGAAGAAGUUACAACAACUCUGGAAGAAACUAAGUUCCUCACAGAAAACUUGUUACUUUAUAUUGACAUUAAUGGCAAUCUUCAUCCAGAUUCUGCCACUCUUGUUAGUGACAUUGACAUCACUUUCUUAAAGAAAGAUGCUCCAUAUAUAGUGGGUGAUGUUGUUCAAGAGGGUGUUUUAACUGCUGUGGUUAUACCUACUAAAAAGGCUGGUGGCACUACUGAAAUGCUAGCGAAAGCUUUGAGAAAAGUGCCAACAGACAAUUAUAUAACCACUUACCCGGGUCAGGGUUUAAAUGGUUACACUGUAGAGGAGGCAAAGACAGUGCUUAAAAAGUGUAAAAGUGCCUUUUACAUUCUACCAUCUAUUAUCUCUAAUGAGAAGCAAGAAAUUCUUGGAACUGUUUCUUGGAAUUUGCGAGAAAUGCUUGCACAUGCAGAAGAAACACGCAAAUUAAUGCCUGUCUGUGUGGAAACUAAAGCCAUAGUUUCAACUAUACAGCGUAAAUAUAAGGGUAUUAAAAUACAAGAGGGUGUGGUUGAUUAUGGUGCUAGAUUUUACUUUUACACCAGUAAAACAACUGUAGCGUCACUUAUCAACACACUUAACGAUCUAAAUGAAACUCUUGUUACAAUGCCACUUGGCUAUGUAACACAUGGCUUAAAUUUGGAAGAAGCUGCUCGGUAUAUGAGAUCUCUCAAAGUGCCAGCUACAGUUUCUGUUUCUUCACCUGAUGCUGUUACAGCGUAUAAUGGUUAUCUUACUUCUUCUUCUAAAACACCUGAAGAACAUUUUAUUGAAACCAUCUCACUUGCUGGUUCCUAUAAAGAUUGGUCCUAUUCUGGACAAUCUACACAACUAGGUAUAGAAUUUCUUAAGAGAGGUGAUAAAAGUGUAUAUUACACUAGUAAUCCUACCACAUUCCACCUAGAUGGUGAAGUUAUCACCUUUGACAAUCUUAAGACACUUCUUUCUUUGAGAGAAGUGAGGACUAUUAAGGUGUUUACAACAGUAGACAACAUUAACC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1 29903 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2 29903 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3 29903 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mismatches X X X X 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positions 1 21 41 61 81 101 121 141 161 181 201 221 241 261 281 301 321 341 361 381 401 421 441 461 481 501 521 541 561 581 601 621 641 661 681 701 721 741 761 781 801 821 841 861 881 901 921 941 961 981 1001 1021 1041 1061 1081 1101 1121 1141 1161 1181 1201 1221 1241 1261 1281 1301 1321 1341 1361 1381 1401 1421 1441 1461 1481 1501 1521 1541 1561 1581 1601 1621 1641 1661 1681 1701 1721 1741 1761 1781 1801 1821 1841 1861 1881 1901 1921 1941 1961 1981 2001 2021 2041 2061 2081 2101 2121 2141 2161 2181 2201 2221 2241 2261 2281 2301 2321 2341 2361 2381 2401 2421 2441 2461 2481 2501 2521 2541 2561 2581 2601 2621 2641 2661 2681 2701 2721 2741 2761 2781 2801 2821 2841 2861 2881 2901 2921 2941 2961 2981 3001 3021 3041 3061 3081 3101 3121 3141 3161 3181 3201 3221 3241 3261 3281 3301 3321 3341 3361 3381 3401 3421 3441 3461 3481 3501 3521 3541 3561 3581 3601 3621 3641 3661 3681 3701 3721 3741 3761 3781 3801 3821 3841 3861 3881 3901 3921 3941 3961 3981 4001 4021 4041 4061 4081 4101 4121 4141 4161 4181 4201 4221 4241 4261 4281 4301 4321 4341 4361 4381 4401 4421 4441 4461 4481 4501 4521 4541 4561 4581 4601 4621 4641 4661 4681 4701 4721 4741 4761 4781 4801 4821 4841 4861 4881 4901 4921 4941 4961 4981 5001 5021 5041 5061 5081 5101 5121 5141 5161 5181 5201 5221 5241 5261 5281 5301 5321 5341 5361 5381 5401 5421 5441 5461 5481 5501 5521 5541 5561 5581 5601 5621 5641 5661 5681 5701 5721 5741 5761 5781 5801 5821 5841 5861 5881 5901 5921 5941 5961 5981 6001 6021 6041 6061 6081 6101 6121 6141 6161 6181 6201 6221 6241 6261 6281 6301 6321 6341 6361 6381 6401 6421 6441 6461 6481 6501 6521 6541 6561 6581 6601 6621 6641 6661 6681 6701 6721 6741 6761 6781 6801 6821 6841 6861 6881 6901 6921 6941 6961 6981 7001 7021 7041 7061 7081 7101 7121 7141 7161 7181 7201 7221 7241 7261 7281 7301 7321 7341 7361 7381 7401 7421 7441 7461 7481 7501 7521 7541 7561 7581 7601 7621 7641 7661 7681 7701 7721 7741 7761 7781 7801 7821 7841 7861 7881 7901 7921 7941 7961 7981 8001 8021 8041 8061 8081 8101 8121 8141 8161 8181 8201 8221 8241 8261 8281 8301 8321 8341 8361 8381 8401 8421 8441 8461 8481 8501 8521 8541 8561 8581 8601 8621 8641 8661 8681 8701 8721 8741 8761 8781 8801 8821 8841 8861 8881 8901 8921 8941 8961 8981 9001 9021 9041 9061 9081 9101 9121 9141 9161 9181 9201 9221 9241 9261 9281 9301 9321 9341 9361 9381 9401 9421 9441 9461 9481 9501 9521 9541 9561 9581 9601 9621 9641 9661 9681 9701 9721 9741 9761 9781 9801 9821 9841 9861 9881 9901 9921 9941 9961 9981 10001 10021 10041 10061 10081 10101 10121 10141 10161 10181 10201 10221 10241 10261 10281 10301 10321 10341 10361 10381 10401 10421 10441 10461 10481 10501 10521 10541 10561 10581 10601 10621 10641 10661 10681 10701 10721 10741 10761 10781 10801 10821 10841 10861 10881 10901 10921 10941 10961 10981 11001 11021 11041 11061 11081 11101 11121 11141 11161 11181 11201 11221 11241 11261 11281 11301 11321 11341 11361 11381 11401 11421 11441 11461 11481 11501 11521 11541 11561 11581 11601 11621 11641 11661 11681 11701 11721 11741 11761 11781 11801 11821 11841 11861 11881 11901 11921 11941 11961 11981 12001 12021 12041 12061 12081 12101 12121 12141 12161 12181 12201 12221 12241 12261 12281 12301 12321 12341 12361 12381 12401 12421 12441 12461 12481 12501 12521 12541 12561 12581 12601 12621 12641 12661 12681 12701 12721 12741 12761 12781 12801 12821 12841 12861 12881 12901 12921 12941 12961 12981 13001 13021 13041 13061 13081 13101 13121 13141 13161 13181 13201 13221 13241 13261 13281 13301 13321 13341 13361 13381 13401 13421 13441 13461 13481 13501 13521 13541 13561 13581 13601 13621 13641 13661 13681 13701 13721 13741 13761 13781 13801 13821 13841 13861 13881 13901 13921 13941 13961 13981 14001 14021 14041 14061 14081 14101 14121 14141 14161 14181 14201 14221 14241 14261 14281 14301 14321 14341 14361 14381 14401 14421 14441 14461 14481 14501 14521 14541 14561 14581 14601 14621 14641 14661 14681 14701 14721 14741 14761 14781 14801 14821 14841 14861 14881 14901 14921 14941 14961 14981 15001 15021 15041 15061 15081 15101 15121 15141 15161 15181 15201 15221 15241 15261 15281 15301 15321 15341 15361 15381 15401 15421 15441 15461 15481 15501 15521 15541 15561 15581 15601 15621 15641 15661 15681 15701 15721 15741 15761 15781 15801 15821 15841 15861 15881 15901 15921 15941 15961 15981 16001 16021 16041 16061 16081 16101 16121 16141 16161 16181 16201 16221 16241 16261 16281 16301 16321 16341 16361 16381 16401 16421 16441 16461 16481 16501 16521 16541 16561 16581 16601 16621 16641 16661 16681 16701 16721 16741 16761 16781 16801 16821 16841 16861 16881 16901 16921 16941 16961 16981 17001 17021 17041 17061 17081 17101 17121 17141 17161 17181 17201 17221 17241 17261 17281 17301 17321 17341 17361 17381 17401 17421 17441 17461 17481 17501 17521 17541 17561 17581 17601 17621 17641 17661 17681 17701 17721 17741 17761 17781 17801 17821 17841 17861 17881 17901 17921 17941 17961 17981 18001 18021 18041 18061 18081 18101 18121 18141 18161 18181 18201 18221 18241 18261 18281 18301 18321 18341 18361 18381 18401 18421 18441 18461 18481 18501 18521 18541 18561 18581 18601 18621 18641 18661 18681 18701 18721 18741 18761 18781 18801 18821 18841 18861 18881 18901 18921 18941 18961 18981 19001 19021 19041 19061 19081 19101 19121 19141 19161 19181 19201 19221 19241 19261 19281 19301 19321 19341 19361 19381 19401 19421 19441 19461 19481 19501 19521 19541 19561 19581 19601 19621 19641 19661 19681 19701 19721 19741 19761 19781 19801 19821 19841 19861 19881 19901 19921 19941 19961 19981 20001 20021 20041 20061 20081 20101 20121 20141 20161 20181 20201 20221 20241 20261 20281 20301 20321 20341 20361 20381 20401 20421 20441 20461 20481 20501 20521 20541 20561 20581 20601 20621 20641 20661 20681 20701 20721 20741 20761 20781 20801 20821 20841 20861 20881 20901 20921 20941 20961 20981 21001 21021 21041 21061 21081 21101 21121 21141 21161 21181 21201 21221 21241 21261 21281 21301 21321 21341 21361 21381 21401 21421 21441 21461 21481 21501 21521 21541 21561 21581 21601 21621 21641 21661 21681 21701 21721 21741 21761 21781 21801 21821 21841 21861 21881 21901 21921 21941 21961 21981 22001 22021 22041 22061 22081 22101 22121 22141 22161 22181 22201 22221 22241 22261 22281 22301 22321 22341 22361 22381 22401 22421 22441 22461 22481 22501 22521 22541 22561 22581 22601 22621 22641 22661 22681 22701 22721 22741 22761 22781 22801 22821 22841 22861 22881 22901 22921 22941 22961 22981 23001 23021 23041 23061 23081 23101 23121 23141 23161 23181 23201 23221 23241 23261 23281 23301 23321 23341 23361 23381 23401 23421 23441 23461 23481 23501 23521 23541 23561 23581 23601 23621 23641 23661 23681 23701 23721 23741 23761 23781 23801 23821 23841 23861 23881 23901 23921 23941 23961 23981 24001 24021 24041 24061 24081 24101 24121 24141 24161 24181 24201 24221 24241 24261 24281 24301 24321 24341 24361 24381 24401 24421 24441 24461 24481 24501 24521 24541 24561 24581 24601 24621 24641 24661 24681 24701 24721 24741 24761 24781 24801 24821 24841 24861 24881 24901 24921 24941 24961 24981 25001 25021 25041 25061 25081 25101 25121 25141 25161 25181 25201 25221 25241 25261 25281 25301 25321 25341 25361 25381 25401 25421 25441 25461 25481 25501 25521 25541 25561 25581 25601 25621 25641 25661 25681 25701 25721 25741 25761 25781 25801 25821 25841 25861 25881 25901 25921 25941 25961 25981 26001 26021 26041 26061 26081 26101 26121 26141 26161 26181 26201 26221 26241 26261 26281 26301 26321 26341 26361 26381 26401 26421 26441 26461 26481 26501 26521 26541 26561 26581 26601 26621 26641 26661 26681 26701 26721 26741 26761 26781 26801 26821 26841 26861 26881 26901 26921 26941 26961 26981 27001 27021 27041 27061 27081 27101 27121 27141 27161 27181 27201 27221 27241 27261 27281 27301 27321 27341 27361 27381 27401 27421 27441 27461 27481 27501 27521 27541 27561 27581 27601 27621 27641 27661 27681 27701 27721 27741 27761 27781 27801 27821 27841 27861 27881 27901 27921 27941 27961 27981 28001 28021 28041 28061 28081 28101 28121 28141 28161 28181 28201 28221 28241 28261 28281 28301 28321 28341 28361 28381 28401 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Compare normalized reactivities between experiments¶
In order to determine the extent of agreement between experimental reactivities, RNAvigate is used to plot sample vs. sample reactivies and to compute Pearson coefficients. Here, a kernel density plot is used, instead of the default scatter plot, which allows better visualization for large data sets.
plot = rnav.plot_linreg(
samples=[lan, huston, manfredonia, sun],
profile="shapemap",
scale='linear',
kde=True,
colorbars=False,
)
for row in plot.axes:
for ax in row:
ax.set(
xlim=(-0.1, 2.6),
ylim=(-0.1, 2.6),
xticks=[0, 0.5, 1.0, 1.5, 2.0, 2.5],
yticks=[0, 0.5, 1.0, 1.5, 2.0, 2.5],
)
# plot.save("pairwise_regression.svg")
Compare structure models¶
These 4 studies ultimately resulted in 4 different structure models, all based on in-cell structure probing experiments which differed at every experimental and computational step. For each structure we compute the percentage of base pairs predicted by 1, 2, 3 or 4 studies. Overall, these studies agree on many base-pairs, despite differences in experimental design.
structure_comparison = rnav.data.StructureCompareMany(
input_data=[
lan.get_data("ss"),
huston.get_data("ss"),
manfredonia.get_data("ss"),
sun.get_data("ss")
],
sequence=lan.get_data("ss"),
)
df = structure_comparison.data
orf1 = df.eval("i > 266 & j < 21555")
percentages = [sum(df.loc[orf1, "Num_structures"] == i)*(i+1) / sum(orf1)
for i in [0, 1, 2, 3]]
import matplotlib.pyplot as plt
plt.pie(
x=percentages,
labels=["No other studies", "1 other study", "2 other studies", "3 other studies"],
colors=structure_comparison._metric["cmap"],
startangle=90,
autopct='%1.1f%%')
plt.title("Percent that appear in other studies")
# plt.savefig("pie_chart.svg")
Text(0.5, 1.0, 'Percent that appear in other studies')
Visualize structure models¶
To visualize these structure models, we employ rnav.plot_arcs() to display arc plots with each base-pair represented as an arc and colored by how many models predict it.
To limit the size of these plots, we only visualize nucleotides 1-3000 and 3001-6000 using the region argument.
Each group choose a different value for the max pairing distance parameter provided to the folding algorithm, which limits the similarity seen between structure models. These limits are displayed on this plot using the Matplotlib interface of the RNAvigate plot. Each dashed line represents the maximum height of an arc reported by one of the studies.
rnav.styles.set_defaults(dpi=900)
for start, end in [[1000, 4000], [4000, 7000]]:
plot = rnav.plot_arcs(
samples=[lan],
labels=["All samples"],
sequence=structure_comparison,
structure=de_novo.get_data("ss"),
interactions=structure_comparison,
panels={"interactions": "top",
"structure": "bottom"},
region=[start, end],
seqbar=False,
colorbars=False,
nt_ticks=(500, 100),
)
ax = plot.axes[0, 0]
ax.set(ylim=(-305, 305))
for pairing_distance in [350, 300, 500, 600]:
ax.plot(
[start, end], [pairing_distance/2, pairing_distance/2],
color="grey", ls="--"
)
plot.set_figure_size(height_ax_rel=0.003, width_ax_rel=0.003)
# plot.save(f"ss_compare_{start}_{end}.svg")
rnav.styles.set_defaults(dpi=140)
cb_plot = plot.plot_colorbars()
# cb_plot.save("ss_compare_colorbar.svg")